The X Factor

Two years ago, a gammaretrovirus called xenotropic murine leukemia virus-like virus (XMRV) was linked to CFS with a high-profile study published in Science by a team of researchers from the Whittemore Peterson Institute (WPI), National Cancer Institute (NCI) and Cleveland Clinic [1]. Today, this study by Vincent Lombardi et al. remains the only one to have shown an association between XMRV and CFS. Research groups in seven countries have tested samples from CFS patients and published 16 papers [2-17] that cast doubt on the original data. Through more than 175 publications about XMRV, researchers have documented a better understanding of its origins, life cycle and other properties, yet no links to human disease have been firmly established.

Recent Studies
While several of the early follow-up studies involving samples from CFS patients were small, used broader criteria to define CFS or had other limitations, two major studies published in May 2011 addressed many of the criticisms. The first was from Clifford Shin et al., published May 4 in the Journal of Virology [13]. Konnie Knox et al. published results in Science on May 31 [14]. Both groups tested samples from CFS patients who had tested positive at WPI, yet neither group found evidence of the virus in those samples or others they tested from well-characterized patients using multiple detection methods. Both studies attracted considerable media attention that kept CFS and XMRV in headlines for most of May and early June.

Another group at NCI led by Tobias Paprotka reported in the same issue of Science [18] that XMRV originated through a laboratory recombination of two mouse viruses in the prostate cancer cell line CWR22Rv1. The recombination event occurred between 1993 and 1996. This evidence makes it unlikely that XMRV will be shown to be the sole cause of CFS, which certainly existed before these dates. The XMRV sequences submitted by WPI to GenBank, the public DNA sequence database, are 98-100 percent identical to VP62, an XMRV clone derived from 22Rv1 and used by WPI and the Cleveland Clinic.

The other piece of evidence weighing against XMRV as a human pathogen are multiple reports of contaminants with mouse DNA in laboratory reagents, supplies and equipment that can show falsely positive results in tests for XMRV and other murine leukemia virus-like viruses (MLVs). Four papers published on Dec. 20, 2010, in Retrovirology [19-22] drew considerable attention to the issue of contamination. The two CFS papers published in May (described above) reported specific sources of contaminants that were encountered in their studies. Information posted to NCI’s website in early June states, “a sample of the XMRV viruses reported in the 2009 article has been cultivated from patient samples and was analyzed at NCI. In contrast to the original findings, the new data suggest it is unlikely that these XMRVs were derived from infected patients. Instead, like the other XMRVs that have been sequenced, they appear to be laboratory contaminants.” Robert Silverman of the Cleveland Clinic told the Chicago Tribune in March 2011 that he was “concerned about lab contamination, despite our best efforts to avoid it.” His lab is conducting additional experiments to test for the possibility. WPI has stated repeatedly that its tests for contaminants have been negative.

By the end of May, doubts about the original report had grown so strong that the editor of Science, Bruce Alberts, requested the authors retract the original paper. They declined to do so, stating it was premature. On May 31, Alberts issued an “Editorial Expression of Concern” [23] that is now tagged to the paper pending the outcome of two large multicenter studies being funded by the National Institutes of Health (NIH) (see below).

Supporting evidence for gammaretrovirus association with CFS has come from two groups who have reported finding sequences consistent with the larger family of gammaretroviruses, but not XMRV specifically. The first team, from the Food and Drug Administration (FDA), NIH and Harvard Medical School, published its findings in August 2010 in the Proceedings of the National Academy of Sciences (Shyh-Ching Lo et al.) [24]. Testing stored samples collected in 1993 for a study looking for mycoplasma, Dr. Lo found 32 of 37 samples from CFS patients positive for MLV sequences. Eight patients from that cohort provided fresh samples and seven of those tested positive again. None of the researchers involved in this study has reported any further data. However, two papers published this summer by groups working independently reported that the sequences obtained from two time points were not consistent with evolutionary changes [25, 26].

The other group, led by Cornell University researcher Maureen Hanson, has presented data at meetings, but its findings have not yet been reported in the literature. The WPI has presented positive data from a study of patients in the U.K., but that has not yet been published, either. News about other unpublished data has circulated through the community and some have charged that positive studies are being unfairly declined by journals, but no specific incidents have been made public.

Clarity Ahead?
To bring greater clarity to the issue, NIH is supporting two studies that involve some of the labs that have published conflicting data.

The first is being coordinated by the National Heart, Lung and Blood Institute and is known as the Blood XMRV Scientific Research Working Group study (SRWG). It is a four-phase study [27] designed to evaluate XMRV detection assays in analytical and clinical samples and to make an initial estimate of XMRV prevalence in blood donors.

Results from Phase I were reported in July 2010 at a meeting of the FDA’s Blood Products Advisory Committee (BPAC). They showed that six participating labs (CDC, Gen-Probe, NCI-Drug Resistance Program, WPI and FDA-Lo and FDA-Hewlett) had comparable sensitivity in their testing methods using coded analytical samples spiked with XMRV.

Phase II results were presented at a BPAC meeting in December 2010. The presentation was repeated days later during a webinar hosted by the Solve ME/CFS Initiative. In Phase II, samples were obtained from four subjects whom WPI indicated were positive for XMRV on multiple occasions and one pedigreed negative control (tested in Phase I). Samples were collected in the same manner and then split into three groups for immediate processing and processing after two days and four days to compare results. Five labs (CDC, Gen-Probe, NCI-Drug Resistance Program, WPI and NCI-Ruscetti) tested the samples under blinded conditions using the assays from Phase I. Results from three labs (CDC, Gen-Probe and NCI-Drug Resistance Program) were all negative. WPI had at least one positive result for three of the four XMRV-positive subjects as well as the negative control. NCI-Ruscetti reported positive results for three of four XMRV-positive subjects, as well as the negative control. The results from WPI and NCI-Ruscetti agreed on two of the XMRV-positive subjects and differed on the other two. One of the aims of Phase II was to determine whether processing time affected testing results; the data indicated that it did not. In spite of the other conflicting data, it was deemed that Phase III would proceed.

This spring, samples were collected from 30 subjects who previously tested positive for either XMRV or MLV sequences, two pedigreed negative controls and 12 blood donor controls. Five analytical controls were included in the panel as well. Eight participating labs (CDC, Gen-Probe, NCI-Drug Resistance Program, WPI, NCI-Ruscetti, FDA-Lo, FDA-Hewlett and Abbott) tested blinded samples according to approved protocols and each has submitted its results to the Blood Systems Research Institute for decoding and analysis. Results will be presented at meetings this fall and submitted to a peer-reviewed journal.

The need for Phase IV (focused on blood donors) will be based on the outcome of Phase III.

The second large study is being sponsored by the National Institute for Allergy and Infectious Diseases. W. Ian Lipkin, M.D., of Columbia’s Center for Infection and Immunity, a renowned pathogen hunter, is coordinating the collection of samples from 100 well-characterized CFS patients and 100 matched controls from four sites around the country. Samples will be processed, blinded and sent to labs at the FDA (Lo), CDC and WPI. Dr. Lipkin will break the code and reconcile the results. Although the study was expected to wrap up before year-end, delays in securing required institutional approvals may push the conclusion of the study into 2012.

Other studies continue as well:

The American Red Cross is collaborating with Gen-Probe and Abbott Laboratories to test samples from 10,000 healthy people in six geographic areas. They will also test samples from 120 recipients of blood donations from more than 4,000 donors. Both donors and recipients will be tested for evidence of XMRV and MLVs. [28]

The NCI has evaluated CFS patients who had been previously tested for XMRV at one of its clinics for a study of the different assays used to detect XMRV.

The FDA’s Center for Biologics Evaluation and Research has conducted a study of the transmission and infection processes of XMRV to address potential safety concerns in cells used to produce vaccines and blood products; results have been submitted for publication.

It remains to be seen whether XMRV will provide the answers to better methods of diagnosis and treatment that were heralded in October 2009. There is no question that the original report of an association has attracted remarkable scientific talent, increased engagement by health agencies and unprecedented awareness of the devastating impact of CFS. No other report among the 5,000+ peer-reviewed articles about CFS has attracted this much attention or such sustained effort to investigate more thoroughly. The debate over XMRV has been polarizing at times, but there is no longer dispute about whether CFS is worthy of scientific endeavor; that, in itself, is progress.